Radiation exposure leads to the development of severe acute inflammatory response and late damage to the lung culminating in delayed fibrosis. Pulmonary endothelium is the locus of early structural and functional changes in irradiation induced lung injury and apoptosis of pulmonary endothelium is a major contributing factor to genesis of acute radiation lung injury and maintenance of delayed responses. Nonetheless, the early molecular pathways that account for irradiation induced pulmonary endothelial cell apoptosis are unknown. We have recently shown that upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL acts as a switch to regulate cyt c's mitochondrial functions. In preliminary data, using ion trap electrospray ionization mass spectrometry based lipidomics for the first time in lung, we show that selective oxidation of CL is an early event in irradiated lung and also occurs in simple models (e.g. LPS) of apoptosis in cultured pulmonary endothelial cells. Furthermore we have designed an antioxidant/electron scavenger (XJB-5-131;an hemigramicidin-4-amino-2,2,6,6-tetramethylpiperidine-N- oxyl) and a peroxidase-activatable nitric oxide donor (HVTP, 2-hydroxylamino-vinyl-triphenyl-phosphonium) that are specifically targeted to mitochondria and show that they can effectively inhibit apoptosis in a variety of cell types and ameliorate acute organ dysfunction in vivo. Accordingly, the SPECIFIC AIMS of this proposal are to determine: 1) the molecular role of cardiolipin-induced peroxidase activity of cytochrome c in pulmonary endothelial cell apoptosis in mouse after lung irradiation;2) the molecular role of oxidized anionic phospholipids in the signal transduction pathway of pulmonary endothelial cell apoptosis including scavenging of apoptotic pulmonary endothelium by macrophages;3) the therapeutic potential of hemigramicidin-4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl, XJB-5-131, and peroxidase-activatable nitric oxide donor, 2-hydroxyamino-vinyl-triphenyl-phosphonium, HVTP, - two types of mitochondria targeted antioxidants/ inhibitors of cytochrome c/cardiolipin peroxidase - in preventing pulmonary endothelial cell cardiolipin oxidation, apoptosis and favorably affecting the course of lung injury and delayed fibrosis after lung irradiation. Antioxidants have been long viewed as potentially effective for therapeutic interventions. Elucidation of specific mechanisms through which cyt c and CL oxidation products link free radical production with early apoptotic events in mitochondria and employment of novel targeting protocols of antioxidants/electron scavengers to both pulmonary endothelium (using immuno-nanoparticles and mitochondria represent a new strategy in drug discovery. PUBLIC HEALTH RELEVANCE: Radiation exposure leads to the development of severe acute inflammatory response and late damage to the lung culminating in delayed fibrosis. Pulmonary endothelium is the locus of early structural and functional changes in irradiation induced lung injury and apoptosis of pulmonary endothelium is a major contributing factor to genesis of acute radiation lung injury and maintenance of delayed responses. Nonetheless, the early molecular pathways that account for irradiation induced pulmonary endothelial cell apoptosis are unknown. The proposed project will elucidate specific mechanisms of early apoptotic events in mitochondria and employ novel targeting protocols of antioxidants/electron scavengers to pulmonary endothelium (using immuno-nanoparticles) and mitochondria that represent a new strategy in drug discovery.